Preclinical Validation of Transglutaminase 2 as a Novel Target for Celiac Disease

描述(由申请人提供)：拟议研究的首要目标是利用最先进的小分子先导化合物和目前可用的最先进的动物模型，在临床前验证转谷氨酰胺酶2(TG2)作为治疗乳糜泻(CED)的药物靶点。CED是一种由面筋引起的终生小肠炎性疾病，到目前为止还没有非饮食疗法。尽管TG2被广泛认为是CED治疗中最有吸引力的小分子药物靶点，但这一药理学假说仍然难以确定。在初步研究中，我们研究了一类基于温和亲电的3-溴-4，5-二氢异恶唑基序的不可逆TG2抑制剂，并鉴定了一种具有良好体内外活性的先导化合物(ERW1041E)。重要的是，ERW1041E是我们所知的唯一一种TG2抑制剂，已被证明在体内每天一次的剂量计划中阻断TG2。另外，通过改造双转基因和三转基因小鼠品系，我们已经建立了一个动物模型，该模型显示了CED的所有四个特征：(I)面筋依赖的T细胞激活；(Ii)面筋依赖的自身抗体的产生；(Iii)面筋依赖的淋巴细胞渗入肠上皮；以及(Iv)面筋依赖的绒毛萎缩。最后但并非最不重要的一点是，我们已经证明，口服面筋暴露可以激活该模型小鼠小肠中的TG2，并可被我们的先导化合物ERW1041E阻断。我们确认TG2作为CED治疗靶点的计划有两个具体目标：1)我们将通过两个重点化合物系列的合成和体外评估来确定ERW1041E的改进类似物。最有希望的类似物将接受药代动力学和初步毒理学分析。特别是，我们寻求在不牺牲其效力、耐受性、口服生物利用度或溶解性的情况下提高ERW1041E的异构体特异性。2)为了验证TG2作为CED药物靶点的有效性，将为ERW1041E和最多四个改进的类似物定义抑制剂剂量与肠道TG2活性之间的药效关系。从这些数据中，将选择每种化合物的次最大和最大抑制剂量，并用于询问TG2在不同临床相关场景中的作用，这些场景可以在我们的小鼠模型中准确模拟。总之，这些研究将为最有希望的候选药物的临床评估提供明确的方向。如果上述研究成功，那么该项目中最有希望的TG2抑制剂可能是在CED患者中进行测试的合适候选者。值得注意的是，最近的努力导致了这类概念验证研究的短期(2周)、小(40名患者)临床方案的发展。因此，在适当的非临床安全性研究之前，CED治疗的新靶点可能在下一年内得到临床验证。 五年了。

DESCRIPTION (provided by applicant): The overarching goal of the proposed studies is to preclinically validate transglutaminase 2 (TG2) as a drug target for celiac disease (CeD), using the most advanced small molecule leads and the most advanced animal models available to date. CeD is a life-long, gluten-induced inflammatory disorder of the small intestine for which no non-dietary therapy exists to date. Although TG2 is widely regarded as the most attractive small molecule drug target for CeD therapy, definitive support for this pharmacologic hypothesis remains elusive. In preliminary studies, we have studied a class of irreversible TG2 inhibitors based on the mildly electrophilic 3- bromo-4,5-dihydroisoxazole motif, and have identified a lead compound (ERW1041E) with good in vitro and in vivo activity. Importantly, ERW1041E is the only TG2 inhibitor to our knowledge that has been shown to block TG2 in vivo on a once-daily dosing schedule. Separately, by engineering double and triple transgenic mouse strains, we have established an animal model that shows all four hallmarks of CeD: (i) gluten-dependent T cell activation; (ii) gluten-dependent autoantibody production; (iii) gluten-dependent infiltrationof lymphocytes into the intestinal epithelium; and (iv) gluten-dependent villous atrophy. Last but not least, we have demonstrated that oral gluten exposure activates TG2 in the small intestine of this mouse model and can be blocked by our lead compound ERW1041E. Our plans for validation of TG2 as a therapeutic target for CeD are described under two Specific Aims: 1) We will identify improved analogs of ERW1041E through synthesis and in vitro evaluation of two focused compound series. The most promising analogs will be subjected to pharmacokinetic and preliminary toxicological analysis. In particular, we seek to improve the isoform specificity o ERW1041E without sacrificing its potency, tolerability, oral bioavailability, or solubility. 2) To validate TG2 as a CeD drug target, the pharmacodynamic relationship between inhibitor dose and intestinal TG2 activity will be defined for ERW1041E and up to four improved analogs. From this data, sub-maximal and maximal inhibitory doses of each compound will be selected, and used to interrogate the role of TG2 in different clinically relevant scenarios that can be accurately mimicked in our mouse models. Together, these studies will provide clear direction for clinical evaluation of the most promising drug candidate. If the above studies are successful, then the most promising TG2 inhibitor from this project may be a suitable candidate for testing in CeD patients. Notably, recent efforts have led to the development of a short (2-week), small (40-patient) clinical protocol for such proof-of-concept studies. Thus, pending appropriate non-clinical safety studies, a new target for CeD therapy could be clinically validated within the next 5 years.